1. Biochemistry Clinical practice Lecturer of Biochemistry
Serum biochemical parameters urea assay
Biochemistry Clinical practice
CLS 432
Dr. Samah Kotb
Lecturer of Biochemistry
2015

2. principle
Serum urea concentration was assayed by using commercial kit, supplied by Biodiagnostics (Egypt), (Fawcett and Scott, 1960).
Enzymatic determination of urea according to the following reaction:

3. principle Urease Urea + H2O 2NH3 + CO2
In an alkaline medium, the ammonium ions react with the phenol and hypochlorite to form a green colored indophenol which absorbs light at 550 nm proportional to initial urea concentration.

4. Buffer- Enzyme reagent
Reagents
Reagent 1
Urea Standard
50 mg/dl
Reagent 2
Buffer- Enzyme reagent
Phosphate buffer
Urease
> 5000 U/l
50 mmol/l
>10000U/l
Reagent 3
Color reagent
Phenol
Sodium -nitroprusside
100 mmol/l
0.2 mmol/l
Reagent 4
Alkaline Reagent
Sodium hydroxide
Sodium hypochlorite
150 mmol/l
15 mmol/l

5. Procedure Blank Standard Sample ---------- 10 µl Reagent 2 0.2 ml
Blank
Standard
Sample
10 µl
Reagent 2
0.2 ml
All tubes were mixed and incubated for 5 minutes at 37 oC
Reagent 3
1.0 ml
Reagent 4

6. All test tubes were mixed and incubated for 10 minutes at 37oC
All test tubes were mixed and incubated for 10 minutes at 37oC . The absorbance of the sample and standard were measured against reagent blank at 550 nm.

7. Calculation:
The concentration of serum urea (mg/dl) =
= (A sample )/(A standard )x 50 (standard conc.)

8. Serum urea normally vary depending on
Age ( due to change kidney function )
Sex ( conc. are slightly higher in men )
Diet ( protein diet Urea )